CN113461815A - Monoclonal antibody for identifying botrytis cinerea and hybridoma cell strain BcA4 thereof - Google Patents
Monoclonal antibody for identifying botrytis cinerea and hybridoma cell strain BcA4 thereof Download PDFInfo
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
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Abstract
The invention discloses a monoclonal antibody for identifying botrytis cinerea and a hybridoma cell strain thereof, wherein the monoclonal antibody for identifying the botrytis cinerea is prepared by the following preservation numbers: CCTCC No: c2021115 hybridoma cell line secretion; the hybridoma cell strain is named as BcA4 and is preserved in China Center for Type Culture Collection (CCTCC) at 2021, 5 months and 13 days, and the preservation number is as follows: CCTCC No: C2021115. the monoclonal antibody is used for identifying animal and plant diseases caused by botrytis cinerea infection, dynamically monitoring and biologically researching botrytis cinerea, and a large amount of monoclonal antibody can be obtained by injecting the cell strain into the abdominal cavity of a Balb/c mouse.
Description
Technical Field
The invention belongs to the field of animals, and particularly relates to a monoclonal antibody for identifying botrytis cinerea and a hybridoma cell strain BcA4 thereof.
Background
The fungus Botrytis cinerea of Ascomycota is one of the pathogenic fungi of Botrytis cinerea. The pathogenic bacteria can be lost in soil with the hypha to live through the winter, and the fritillaria is infected again in the next year. Botrytis cinerea is also the pathogenic bacterium of botrytis of numerous crops, and causes serious loss to agricultural production every year. The sclerotinia fungi are various in species, and the shapes of colonies, hyphae and spores of related species are similar, so that the fungi are difficult to distinguish by means of the characteristics. The monoclonal antibody (monoclonal antibody) combined with enzyme-linked immunosorbent assay (ELISA) for botrytis cinerea disclosed by the invention has the characteristics of high sensitivity and strong specificity, and is suitable for detecting a large amount of field botrytis cinerea, so that a foundation is laid for identification and biological research of botrytis cinerea by using the antibody and dynamic monitoring of diseases infected by botrytis cinerea, such as gray mold of thunberg fritillary bulb.
Disclosure of Invention
The invention provides a monoclonal antibody for identifying botrytis cinerea, which is prepared from the following components in parts by weight: CCTCC No: c2021115 is secreted.
Furthermore, the invention also provides a hybridoma cell strain which is classified and named as BcA4 and is preserved in China Center for Type Culture Collection (CCTCC) at 5 months and 13 days of 2021, and Wuhan university in Wuhan City, China with the preservation number as follows: CCTCC No: C2021115.
the invention has the following beneficial effects:
(1) the monoclonal antibody has strong specificity: the monoclonal antibody has strong reaction with botrytis cinerea antigen, does not react with alternaria alternate, alternaria tenuissima, fusarium oxysporum, fusarium solani, fusarium equiseti, fusarium semitectum, phoma and phomopsis antigen, and can be used for detecting botrytis cinerea and thunberg fritillary gray mold initiated by the botrytis cinerea.
(2) The detection sensitivity of the monoclonal antibody is high: the indirect ELISA detection result shows that the detection sensitivity of the monoclonal antibody to the antigen prepared from the botrytis cinerea hyphae and spores is 39.06ng/mL (namely 3.906ng per well), and the monoclonal antibody has good development and application prospects.
(3) The monoclonal antibody is Ig lambda.
(4) The monoclonal antibody-bound target protein is single: the monoclonal antibody only binds to an antigen protein of about 25kDa of Botrytis cinerea.
Drawings
FIG. 1 is a potency assay graph of BcA 4;
FIG. 2 reaction profile of BcA4 with different fungal antigens;
FIG. 3 is a graph showing the detection sensitivity measurement of BcA 4;
FIG. 4 is a graph depicting type and subclass identification of BcA4 antibodies;
FIG. 5 is a graph of a target protein of Botrytis cinerea bound by the BcA4 antibody.
Detailed Description
The invention is further explained below with reference to the figures and examples.
Example 1: preparation of hybridoma cell line
(1) Preparation of antigen: selecting single colonies of botrytis cinerea, respectively inoculating the single colonies into a potato glucose liquid culture medium, carrying out shake culture at 25 ℃ for 4-5 days, collecting spores and hyphae by using a 50mL centrifuge tube, centrifuging at 6000r/min for 20min, washing for 2 times by using PBS, carrying out ultrasonic crushing (power 200W, crushing for 2s and interval for 2s), centrifuging the crushed liquid at 6000r/min for 20min, collecting supernatant, measuring the content of albumin by using a Coomassie brilliant blue method, adjusting the protein concentration to 1000 mu g/mL, serving as an initial antigen for immune antigen and later detection, subpackaging a small amount of antigen liquid, and freezing and storing at-80 ℃. A small amount of the extract is stored at-20 deg.C before use.
(2) 3 healthy BaL b/c mice of 6-8 weeks are selected, and an intraperitoneal injection method is adopted, wherein 200 mu L of antigen emulsified by equivalent Freund's complete adjuvant is injected into each mouse. The immunization was carried out 3 weeks later with 200. mu.L of antigen emulsified with an equal amount of Freund's incomplete adjuvant. After another 3 weeks, immunization was performed with antigen without adjuvant. 3 days after the last immunization, antisera (polyclonal antibodies) were collected and mouse spleen lymphocytes were taken under sterile conditions for cell fusion.
(3) Polyethylene glycol (PEG-4000) was used as the fusogenic agent, and the mouse myeloma cell line was SP 2/0. The whole process is operated under aseptic conditions. Placing mouse splenocytes and myeloma cells into 50mL centrifuge tube, mixing, centrifuging (1200r/min, 2min), and completely suckingThe supernatant was homogenized with the fingers. The centrifuge tube was placed in a water-containing beaker pre-warmed in a 37 ℃ water bath, and 0.7mL of a cell fusion agent (50% PEG, pH adjusted to 9.0) was slowly added over 1 min. After standing for 1min, 40mL of RMPI-1640 medium, which had been pre-warmed to 37 ℃, was gradually added to dilute the PEG and lose its effect. After centrifugation (1000r/min, 2min), the supernatant was discarded. The precipitated cells were suspended in 40mL of HAT medium, and then distributed in 96-well cell culture plates previously supplemented with feeder cells, and subjected to 5% (V/V) CO2Incubate at 37 ℃ in an incubator. Half of HAT culture medium is changed after 3 days, half of HAT culture medium is changed after 5 days, and HT culture medium is used for continuous culture after 5 days, at the moment, parents die, and if cells grow, the hybridoma cells are obtained.
(4) When the cells in the small holes grow to cover one fourth of the area of the bottom of the holes, the supernatant can be sucked up and the antibody can be detected by indirect ELISA. Selecting strong positive cell strains which do not react with antigens of fusarium equiseti, fusarium semitectum, alternaria alternata, alternaria tenuis, fusarium oxysporum, fusarium solani, phoma phomopsis and phomopsis, performing multiple cloning culture by using a limiting dilution method until all the holes are positive to obtain a cell strain BcA4 secreting monoclonal antibodies, and further performing expanded culture on the cell strain BcA4 for preparing monoclonal antibody ascites and freezing and storing by liquid nitrogen. The hybridoma cells are preserved in China Center for Type Culture Collection (CCTCC) at 2021, 5 months and 13 days, and the preservation numbers of Wuhan university in Wuhan City are as follows: CCTCC No: C2021115.
example 2: generation of monoclonal antibodies
Taking about 8 weeks old Balb/c mice, performing intraperitoneal injection of 0.2mL of pristanane, and performing intraperitoneal injection of 0.2mL of pristanane containing 5-10 × 10 after 7 days5And (3) carrying out cell suspension on the hybridoma cells, observing the obvious expansion of the abdominal cavity of the mouse 7-10 days after injection, taking ascites, centrifuging at 2000r/min for 3min, and collecting supernatant, namely the ascitic monoclonal antibody. Purifying monoclonal antibody by Protein A column chromatography, and storing at-80 deg.C. The monoclonal antibody is prepared from the BcA4 cell strain, namely the monoclonal antibody which can specifically identify the botrytis cinerea.
Example 3: potency assay for monoclonal antibodies
The titer of the antibody was determined by indirect ELISA. The botrytis cinerea with the concentration of 1000 mu g/mL is used for resistingThe original solution is diluted 500 times with coating solution, coated on a whole piece of enzyme label plate (2 mug/mL), kept overnight at 4 ℃ to be adsorbed on polystyrene plate holes, washed three times by PBST and then sealed by skimmed milk for 60 min. Diluting monoclonal antibody BcA4 at a multiple ratio, adding 100 μ L of the diluted monoclonal antibody into each well, washing at 37 deg.C for 1H, washing PBST for three times, adding 100 μ L of the diluted monoclonal antibody (Sigma) diluted 5000 times according to the instruction, washing at 37 deg.C for 1H, adding OPD substrate developer to develop color, and developing with 50 μ L of 2M H2SO4After the reaction was terminated, the OD was read with a microplate reader490nmAnd determining the titer of the monoclonal antibody ascites by taking the positive result that the negative ratio is more than 2.
Through detection, the titer of the BcA4 is determined to be 1.28 multiplied by 10 dilution4Double, a 1000-fold dilution of BcA4 was determined for other detection experiments (see fig. 1).
Example 4: specificity detection experiment of monoclonal antibody
The detection objects are respectively botrytis cinerea, fusarium equiseti, fusarium semitectum, alternaria tenuis, fusarium oxysporum, fusarium solani, phoma and phomopsis, and the antigens (1000 mu g/mL) are diluted to 2 mu g/mL by using a coating solution and then coated on an enzyme label plate. And (3) detecting the specificity of the monoclonal antibody by using an indirect ELISA method by using the coating solution as a blank control, wherein the monoclonal antibody is diluted by 1000 times, and the enzyme-labeled secondary antibody is diluted by 5000 times.
The detection shows that the antibody and the antigen of the fungus do not have cross reaction, but the antibody has strong reaction with the botrytis cinerea antigen, and the existence of the botrytis cinerea antigen can be obviously judged (figure 2).
Example 5: sensitivity detection experiment of monoclonal antibody
The sensitivity of the monoclonal antibody is determined by an indirect ELISA method. Diluting 1000 μ g/mL botrytis cinerea antigen with carbonate coating solution at a multiple ratio of 1:100 × 20～1:80×214And the enzyme is coated on an enzyme label plate, and the concentration is from top to bottom from high to low. Repeat 8 times, column 12 blank control, only coated with coating solution. The monoclonal antibody is diluted 1000 times, and the enzyme-labeled secondary antibody is diluted 5000 times.
The maximum dilution multiple of the 1000 mug/mL botrytis cinerea antigen obtained by detection is 25600 times, namely the detection sensitivity is as follows: 1000 μ g/mL/25600 ═ 39.06ng/mL, i.e. 3.906ng per well (fig. 3).
Example 6: type and subclass detection experiment of monoclonal antibody
The monoclonal antibody was identified using a mouse monoclonal antibody subclass identification kit (Baiotton center for laboratory materials, lot: 20200809). 1000. mu.g/mL of Botrytis cinerea antigen diluted 1000-fold with the coating solution was applied to an ELISA plate at 100. mu.L/well, 16 wells were repeated, incubated at 37 ℃ for 2h or placed in a4 ℃ apparatus for 12h, and washed 1 time with PBST after the coating solution was removed. Adding 100 μ L of monoclonal antibody diluted 1000 times with PBST into each well of ELISA plate, incubating at 37 deg.C for 30min, washing with PBST for 5 times, taking 100 μ L of each enzyme-labeled antibody of 8 detection antibody Ig types and subclasses in the kit, adding into each well, repeating each enzyme-labeled antibody for 2 times, incubating at 37 deg.C for 30min, washing with PBST for 5 times, adding TMB color developing solution, developing for 15min in dark place, and adding 50 μ L of 2M H2SO4The reaction was terminated. Detection OD of enzyme-linked immunosorbent assay (OD)450nmThe type and subclass of the antibody Ig corresponding to the positive hole are the type and subclass of the antibody of the monoclonal antibody.
The antibody type of the BCA4 was determined to be Ig lambda by detection (FIG. 4).
Example 7: detection assay for monoclonal antibody binding proteins
The detection of the antibody binding protein by SDS-PAGE and Western blot technique includes the following steps:
(1) 5% (M/V) of the concentrated gel and 10% (M/V) of the separation gel were prepared.
(2) 80 mu L of botrytis cinerea antigen and 20 mu L of 5 multiplied sample buffer solution are mixed in a 1.5mL centrifuge tube, and are immediately placed in an ice bath for 3min after being subjected to metal bath at 99 ℃ for 10min, and then are centrifuged for 5min at 10000 r/min.
(3) Samples were added to the sample wells with a microsyringe, 2 wells for each sample, and protein Marker was added simultaneously.
(4) Electrophoresis: and (3) firstly keeping the constant voltage of 80V, changing the constant voltage of 120V after the bromophenol blue indicator enters the separation gel, and stopping electrophoresis when the bromophenol blue indicator moves to about 1cm of the lower opening of the gel plate.
(5) Dividing the gel into two parts, soaking one half into staining solution, staining for 45min on a shaking table, and decolorizing with decolorizing solution until the background is clear.
(6) And transferring the other half of the gel to a nitrocellulose membrane by a semi-dry transfer method at a constant pressure of 25V for 30 min.
(7) The transferred membrane was washed 4 times with PBST for 5min each.
(8) The membrane was immersed in 3% (M/V) skim milk and sealed at room temperature for 1 h.
(9) The membrane was immersed in a 2000-fold diluted monoclonal antibody solution in 3% (M/V) skim milk, shaken at 75r/min for 1h at room temperature, and incubated overnight at 4 ℃.
(10) Washing with the step (7).
(11) The membrane was immersed in 8000-fold dilution of horseradish peroxidase-labeled antibody with 3% (M/V) BSA and shaken at room temperature for 1h at 75 r/min.
(12) Washing with the step (7).
(13) The membrane was immersed in 10mL of a freshly prepared DAB color developing solution, slowly shaken away from light until color development was achieved, and the color development reaction was stopped with distilled water. The band developed on the membrane is the specific protein combined by the antibody, and the relative molecular weight of the combined protein is calculated according to the protein Marker.
The antibody was detected to specifically bind to a protein with a molecular weight of about 25kDa from Botrytis cinerea (FIG. 5).
Finally, it should also be noted that the above list is only a specific implementation example of the present invention. It is obvious that the invention is not limited to the above examples of facts, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (2)
1. A monoclonal antibody for identifying botrytis cinerea is prepared from the following components in a preservation number of CCTCC No: c2021115 is secreted.
2. A hybridoma cell strain is classified and named as BcA4 and is preserved in China Center for Type Culture Collection (CCTCC) at 5 months and 13 days of 2021, wherein the preservation numbers are as follows: CCTCC No: C2021115.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110219478A1 (en) * | 2006-07-28 | 2011-09-08 | The Governors Of The University Of Alberta | Recombinant antibodies to sclerotinia antigens |
| WO2013063613A2 (en) * | 2011-10-28 | 2013-05-02 | University Of Maryland | Methods and compositions related to intracellular neutralization by igg |
| CN110804094A (en) * | 2019-12-03 | 2020-02-18 | 浙江中医药大学 | Monoclonal antibody for identifying alternaria tenuis and hybridoma cell strain AtD2 thereof |
| CN110894234A (en) * | 2019-11-29 | 2020-03-20 | 浙江中医药大学 | Monoclonal antibody capable of recognizing alternaria and hybridoma cell strain AaC5 thereof |
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| US20110219478A1 (en) * | 2006-07-28 | 2011-09-08 | The Governors Of The University Of Alberta | Recombinant antibodies to sclerotinia antigens |
| WO2013063613A2 (en) * | 2011-10-28 | 2013-05-02 | University Of Maryland | Methods and compositions related to intracellular neutralization by igg |
| CN110894234A (en) * | 2019-11-29 | 2020-03-20 | 浙江中医药大学 | Monoclonal antibody capable of recognizing alternaria and hybridoma cell strain AaC5 thereof |
| CN110804094A (en) * | 2019-12-03 | 2020-02-18 | 浙江中医药大学 | Monoclonal antibody for identifying alternaria tenuis and hybridoma cell strain AtD2 thereof |
Non-Patent Citations (2)
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| R. BOSSI等: "Development of a monoclonal antibody-based immunodetection assay for Botrytis cinerea", PLANT PATHOLOGY, vol. 41, no. 4, pages 473 * |
| 张银志等: "灰霉菌多克隆抗体的制备与鉴定", 食品与生物技术学报, no. 6, pages 82 - 85 * |
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